Prior to 1997, Recommended Dietary Allowances (RDAs) for vitamin C ascorbic acid), as well as other water-soluble vitamins, were based on preventing deficiency with a margin of safety. We proposed that new RDAs for vitamins, with vitamin C as a model, should be based on concentration-dependent vitamin functions, especially molecular and clinical functions. We termed this overall concentration-function approach in situ kinetics, with both molecular and clinical goals. Principles of in situ kinetics were adopted and expanded by the National Academy of Sciences as part of revised recommendations for all vitamin intakes, first released in 1997 and continuing through 2001. [unreadable] [unreadable] Molecular goals of in situ kinetics are to determine vitamin-specific functions in relation to vitamin concentrations, using biochemical and molecular techniques. Vitamin C function is investigated in human cells such as fibroblasts and neutrophils. To learn how intracellular concentration is regulated, two pathways of vitamin C accumulation were characterized. In the first pathway, vitamin C is transported as such by two carriers that are sodium-dependent, saturable, energy dependent, and inhibited by laboratory-synthesized ascorbate analogs. The two human transporters SVCT1 and SVCT2 were cloned and characterized, and genomic properties and nucleotide polymorphisms were described. Created mice deficient in the transporter SVCT2 did not survive the prenatal period and had very low or undetectable vitamin C concentrations in many but not all tissues, indicating that vitamin C as such is the dominant transported species. Mice have also been created that are deficient in the transporter SVCT1. These mice survive normally, and characterization of them is underway. The second pathway of vitamin C accumulation is dependent on the extracellular oxidized form of vitamin C, dehydroascorbic acid, which is accumulated by the process of ascorbate recycling. In this process, oxidants from cells such as neutrophils oxidize extracellular vitamin C locally to dehydroascorbic acid. Dehydroascorbic acid is transported by facilitative glucose transporters GLUT I, III, and IV, and immediately reduced intracellularly to vitamin C by glutaredoxin (thioltransferase). Glutaredoxin from neutrophils was isolated, identified as the reducing activity, cloned, and characterized. Our studies show that vitamin C recycling occurs in neutrophils when activated by bacteria, and only in neutrophils and not in bacteria. Studies are ongoing to characterize potential roles of vitamin C recycling in neutrophils, including oxidant quenching, bacterial killing, and regulation of neutrophil apoptosis. Studies are in progress to determine proline hydroxylation as a function of vitamin C concentrations in normal human fibroblasts. To date, these findings show that vitamin C function can be determined in relation to its concentration in living cells. Ascorbate analogs have been developed and tested, utilizing expression transport systems, and are specific only for the SVCT and not the GLUT pathway. [unreadable] [unreadable] Clinical goals of in situ kinetics are to characterize concentrations achieved in humans as a function of dose, mechanisms that control these concentrations, and functional consequences. To investigate dose concentration relationships, clinical studies were undertaken in healthy men and women inpatients hospitalized at the NIT Clinical Center for 5-7 months. For the first time, the following were described: the relationship between vitamin C doses over a wide range and its concentrations in plasma and tissues; true bioavailability of vitamin C; vitamin urinary excretion in relation to dose; functional antioxidant consequences of vitamin C in vivo; a three component pharmacokinetics model of vitamin C distribution in humans; and potential adverse effects in relation to dose. Continuing analyses of the extensive data generated from these studies are ongoing. [unreadable] [unreadable] Analyses of the clinical data lead to the concept of tight control. Orally ingested vitamin C over a very wide dose range resulted in tightly controlled plasma and tissue concentrations. Tight control is mediated by at least three coordinated processes: intestinal absorption; tissue transport; and renal filtration coupled to reabsorption. [unreadable] [unreadable] There are major implications of tight control of vitamin C. First, tight control may allow paracrine function, to facilitate local higher concentrations of secreted vitamin C. On-going clinical studies show that ACTH induces paracrine release of ascorbic acid from human adrenal glands. These are first data for any vitamin in mammals showing that vitamin local secretion occurs via endocrine signaling. Second, tight control may be lost if any of the mechanisms responsible are aberrant in disease, especially renal filtration and reabsorption. Clinical and basic studies to explore these possibilities are ongoing, utilizing healthy subjects, subjects with diabetes, and subjects with Fabry Disease. Third, tight control prevents higher concentrations from occurring, because these may have adverse consequences. [unreadable] [unreadable] From these data, we recognized that tight control is transiently bypassed by intravenous administration of ascorbic acid. For several hours after intravenous administration in humans, pharmacologic concentrations of ascorbate persist until they are cleared by renal filtration and excretion. Use of ascorbic acid intravenously, as a drug and not as a nutrient, may have unexpected and exciting implications for cancer treatment and infections. Mechanism studies to explore these possibilities were undertaken in vitro. They showed that ascorbic acid in pharmacologic concentrations, easily achieved in humans by intravenous administration, selectively kills some cancer cells but not normal cells. The mechanism is dependent on extracellular ascorbic acid, its oxidation to ascorbate radical, and protein-dependent formation of hydrogen peroxide. [unreadable] [unreadable] From these in vitro data, we proposed that pharmacologic ascorbic acid is a pro-drug for preferential steady state formation of ascorbate radical and hydrogen peroxide in the extravascular space but not in blood. This hypothesis is now being testing in vivo. Case studies imply that there could be utility of intravenous ascorbic acid in cancer treatment, and a phase I trial just completed with collaborators at McGill University indicate no toxicity. [unreadable] [unreadable] Based on the portions of the clinical data, RDAs for vitamin C in the U.S. and Canada were revised upward in 2000 by the National Academy of Sciences and were also increased in the following countries: Germany, Austria, Denmark, France, Japan, and China. Because health benefits from vitamin C are coupled to ingestion of fruits and vegetables containing the vitamin, we recommend that vitamin C intake is from at least 5 servings of fruits and vegetables daily. Intravenous ascorbic acid as a therapeutic modality in treatment of some cancers has promise. A phase I-II clinical trial of ascorbic acid in patients with selected cancers is in preparation with external collaborators.